Gene therapy of neural cell injuries in vitro using the hypoxia-inducible GM-CSF expression plasmids and water-soluble lipopolymer (WSLP).
Non-viral polymeric gene carriers have been widely investigated but no promising biocompatible polymer was developed for the gene therapy of neural system injuries yet. This study evaluated the potential usage of water-soluble lipopolymer (WSLP) as a gene delivery vehicle in neural lineage cells of SK-N-BE(2)C, a neuroblastoma cell line and primary culture of mouse neural progenitor cells (mNPCs). When tested with the luciferase reporter (pSV-Luc), WSLP showed higher gene transfection efficiency by more than 8-10 folds yet with lower cytotoxicity than polyethylenimine of 1800 Da (PEI1800), a parental polymer, and Lipofectamine 2000. The optimum N/P ratios were 40:1 for WSLP and 10:1 for PEI1800, respectively. The transfection efficiency for both of WSLP and PEI1800 was higher overall in SK-N-BE(2)C cells than in mNPCs. WSLP was also used successfully for the delivery and hypoxia-inducible expression of luciferase reporter plasmid containing the erythropoietin (Epo) enhancer (pEpo-SV-Luc) or RTP801 promoter (pRTP801-Luc). The hypoxia-inducible system and WSLP were then successfully applied to the delivery of granulocyte macrophage colony-stimulating factor (GM-CSF) gene that was previously shown to have neuroprotective effect on neural cell death in vitro and in rat SCI model. The hypoxia-inducible GM-CSF plasmids (pEpo-SV-GM-CSF and pRTP801-GM-CSF) showed induced expression of GM-CSF under hypoxia and decrease in the hypoxia-induced cell death in SK-N-BE(2)C cells. In conclusion, this study demonstrated that WSLP could be an efficient gene delivery carrier for neural cells and gene therapy of GM-CSF using the hypoxia-inducible system could be a potential therapeutic intervention for neural injuries. Further studies are necessary to confirm the current findings in animal models of CNS injuries.
Kim
Jin-Mo
JM
Department of Physiology, Inha University College of Medicine, Incheon, South Korea.
Lee
Minhyung
M
Kim
Kil Hwan
KH
Ha
Yoon
Y
Choi
Jung Kyoung
JK
Park
So Ra
SR
Park
Hyeonseon
H
Park
Hyung Chun
HC
Ahn
Cheol-Hee
CH
Kim
Sung Wan
SW
Choi
Byung Hyune
BH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004921:Erythropoietin / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D011094:Polyethyleneimine / Q000031:analogs & derivatives* / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection / Q000379:methods
D008055:Lipids; C086724:Lipofectamine; C438935:poly(ethylenimine)-co-(N-(2-aminoethyl) ethyleneimin)-co-N-(N-cholesteryloxycarbonyl-(2-aminoethyl)ethylenimine); D004921:Erythropoietin; D049452:Green Fluorescent Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011094:Polyethyleneimine; D008156:Luciferases; D019311:Staurosporine